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Journal of Molecular Biology Dec 2023To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles....
To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.
Topics: Humans; Catalytic Domain; DNA Primase; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Multifunctional Enzymes; Nucleotides
PubMed: 37923120
DOI: 10.1016/j.jmb.2023.168338 -
Viruses Oct 2022Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa...
Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.
Topics: DNA Primase; Cryoelectron Microscopy; Vaccinia virus; DNA Helicases; DNA; DNA Replication; Nucleotides
PubMed: 36298761
DOI: 10.3390/v14102206 -
BioRxiv : the Preprint Server For... May 2023The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA...
The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.
PubMed: 37205351
DOI: 10.1101/2023.05.03.539257 -
Cancers Jul 2022p53 is a common tumor suppressor, and its mutation drives tumorigenesis. What is more, p53 mutations have also been reported to be indicative of poor prognosis in lung...
p53 is a common tumor suppressor, and its mutation drives tumorigenesis. What is more, p53 mutations have also been reported to be indicative of poor prognosis in lung cancer, but the detailed mechanism has not been elucidated. In this study, we found that DNA primase subunit 2 (PRIM2) had a high expression level and associated with poor prognosis in lung cancer. Furthermore, we found that PRIM2 expression was abnormally increased in lung cancer cells with p53 mutation or altered the p53/RB pathway based on database. We also verified that PRIM2 expression was elevated by mutation or deletion of p53 in lung cancer cell lines. Lastly, silence p53 increased the expression of RPIM2. Thus, these data suggest that PRIM2 is a cancer-promoting factor which is regulated by the p53/RB pathway. The p53 tumor-suppressor gene integrates numerous signals that control cell proliferation, cell cycle, and cell death; and the p53/RB pathway determines the cellular localization of transcription factor E2F, which regulates the expression of downstream targets. Next, we explored the role of PRIM2 in lung cancer and found that knockdown of PRIM2 induced cell cycle arrest, increased DNA damage, and increased cell senescence, leading to decreased lung cancer cell proliferation. Lastly, the positive correlation between PRIM2 and E2F/CDK also indicated that PRIM2 was involved in promoting cell cycle mediated by p53/RB pathway. These results confirmed that the expression of PRIM2 is regulated by the p53/RB pathway in lung cancer cells, promotes DNA replication and mismatch repair, and activates the cell cycle. Overall, we found that frequent p53 mutations increased PRIM2 expression, activated the cell cycle, and promoted lung cancer progression.
PubMed: 35884433
DOI: 10.3390/cancers14143370 -
Communications Biology Mar 2021The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by...
The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.
Topics: Cell Nucleus; DNA Polymerase I; DNA Primase; DNA Replication; HEK293 Cells; Humans; Mitosis; Multiprotein Complexes; Mutation; Protein Binding; Telomere; Telomere Homeostasis; Telomere-Binding Proteins
PubMed: 33731801
DOI: 10.1038/s42003-021-01845-4 -
Journal of Molecular Biology Sep 2021Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis....
Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.
Topics: Bacillus subtilis; Bacterial Proteins; DNA Primase; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Guanosine Pentaphosphate; Guanosine Triphosphate
PubMed: 34389317
DOI: 10.1016/j.jmb.2021.167189 -
Nucleic Acids Research Nov 2022DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during...
DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.
Topics: Humans; DNA Polymerase I; Mutagens; DNA Replication; DNA Primase; Mutagenesis; DNA; DNA Primers; RNA
PubMed: 36454017
DOI: 10.1093/nar/gkac1101 -
Scientific Reports Oct 2021PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in...
Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast.
PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3'-GTCG-5' DNA sequence, where the 3'-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Nucleus; DNA; DNA Damage; DNA Primase; DNA Repair; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Mitochondria; Multifunctional Enzymes
PubMed: 34663822
DOI: 10.1038/s41598-021-00151-7 -
BMC Plant Biology Oct 2023The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically...
BACKGROUND
The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).
RESULTS
Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.
CONCLUSIONS
We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
Topics: Animals; DNA Primase; DNA Helicases; DNA-Directed DNA Polymerase; Arabidopsis; Mitochondria; Zinc Fingers; Ribonucleotides; DNA Replication; Bacteriophage T7
PubMed: 37803262
DOI: 10.1186/s12870-023-04477-4 -
Nucleic Acids Research Apr 2024It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the...
It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.
Topics: Tumor Suppressor Protein p53; DNA-Directed DNA Polymerase; Humans; DNA Damage; DNA Replication; DNA Polymerase iota; Proliferating Cell Nuclear Antigen; DNA Repair; Nucleotidyltransferases; DNA-Binding Proteins; Multifunctional Enzymes; DNA Primase; DNA Damage Tolerance
PubMed: 38321962
DOI: 10.1093/nar/gkae061